The Journal of Published by the International Society of Eukaryotic Microbiology Protistologists

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE Ptolemeba n. gen., a Novel Genus of Hartmannellid Amoebae (Tubulinea, Amoebozoa); with an Emphasis on the Taxonomy of Saccamoeba

Pamela M. Watsona, Stephanie C. Sorrella & Matthew W. Browna,b

a Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi, 39762 b Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University, Mississippi State, Mississippi, 39762

Keywords ABSTRACT 18S rRNA; amoeba; amoeboid; Cashia; cristae; freshwater amoebae; Hartmannella; Hartmannellid amoebae are an unnatural assemblage of amoeboid organisms mitochondrial morphology; SSU rDNA; SSU that are morphologically difficult to discern from one another. In molecular phy- rRNA; terrestrial amoebae; tubulinid. logenetic trees of the nuclear-encoded small subunit rDNA, they occupy at least five lineages within Tubulinea, a well-supported clade in Amoebozoa. The Correspondence polyphyletic nature of the hartmannellids has led to many taxonomic problems, M.W. Brown, Department of Biological in particular paraphyletic genera. Recent taxonomic revisions have alleviated Sciences, Mississippi State University, some of the problems. However, the genus Saccamoeba is paraphyletic and is Mississippi State, MS 39762, USA still in need of revision as it currently occupies two distinct lineages. Here, we Telephone number: +1 662-325-2406; report a new clade on the tree of Tubulinea, which we infer represents a novel FAX number: +1 662-325-7939; genus that we name Ptolemeba n. gen. This genus subsumes a clade of hart- e-mail: [email protected] mannellid amoebae that were previously considered in the genus Saccamoeba, but whose mitochondrial morphology is distinct from Saccamoeba. In accor- Received: 6 March 2014; revised 7 May dance with previous research, we formalize the clade as distinct from Sacc- 2014; accepted May 8, 2014. amoeba. Transmission electron microscopy of our isolates illustrate that both molecularly discrete species can be further differentiated by their unique mito- doi:10.1111/jeu.12139 chondrial cristal morphology.

HARTMANNELLIDAE Volkonsky, 1931 is a family of limax- Dykova et al. 2008). The hartmannellids have recently shaped amoebae within Tubulinea of Amoebozoa (Adl been reviewed and redefined (Brown et al. 2011; Smirnov et al. 2012). The family classically includes the genera et al. 2011). Hartmannella, Cashia, Glaeseria, and Saccamoeba (Page The two most speciose hartmannellid genera are Hart- 1974, 1988). These genera are morphologically difficult to mannella and Saccamoeba; however, both genera appear ascertain between taxa, and recent molecular data have to be paraphyletic in molecular phylogenetic reconstruc- proven that the lines between the genera are particularly tions (Dykova et al. 2008). The fundamental difference skewed. The most challenging aspect is that hartmannel- between the two genera is that Saccamoeba lacks a lids look very similar to one another in terms of gross mor- distinctive hyaline cap on its monotactic pseudopodium, phology. Generally, the trophozoites are elongate with a unlike the distinctive hyaline cap observed in Hartmannella high length to breadth of ratio, typically > 4 (Page 1988). (Page 1988). Hartmannella has been recently redefined They most often move in a monotactic fashion, with non- according to molecular phylogenetics in Brown et al. monotactic pseudopodial extensions resulting in abrupt (2011) and is beginning to be taxonomically resolved changes in direction that can be somewhat eruptive in (Smirnov et al. 2011). However, Saccamoeba is still fashion. Cells are uninucleate with a vesicular nucleolus. greatly in need of revision. The first molecular assignation In addition, molecular phylogenetic trees have shown that of Saccamoeba was in 2000 from an isolate earlier identi- even though the hartmannellids are superficially alike at fied at the American Type Culture Collection (ATCC) as the morphological level they are polyphyletic, comprising Saccamoeba limax (ATCC 30942) (Amaral Zettler et al. at least four different clades in nuclear-encoded small 2000). Dykova et al. (2002)isolated an amoeba (strain subunit rDNA (SSU rDNA) trees (Brown et al. 2011; LOS7N) that is nearly identical in SSU rDNA sequence

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–9 1 Ptolemeba n. gen., a New Hartmannellid Genus Watson et al.

(99.58% uncorrected pairwise distance [upd]) to ATCC 0.75 g K2HPO4, 15.0 g agar, and 1 liter ddH2O) Petri 30942, S. limax sensu (Amaral Zettler et al. 2000). They plates. The plates were then incubated for 4 d and examined strain LOS7N under transmission electron observed with a 10X objective under a compound micro- microscopy (TEM) and found that it has unique tubular scope. On the edges of the absorbed water droplet amoe- mitochondrial cristae that are arranged in an unusual heli- bae were observed. For all strains, a single amoeba cell cal pattern that is very uniform (Dykova et al. 2002). This was isolated using a flame sterilized 30-gage platinum cristal configuration is clearly dissimilar to previous TEM wire micro loop, where the loop was used to drag a single reports of S. limax sensu Page, 1985; which has branching amoeba along the agar surface to isolate it away from all tubular cristae that do not appear to have a uniform other eukaryotes on the inoculated plate. Each culture arrangement (Page 1985). Later, Dykova et al. (2008) iso- was established as clonal with no other accompanying lated a new strain (NTSHR) assigned to Saccamoeba that eukaryotes. Cultures are maintained through monthly pas- appears to be very similar to Page’s concept of S. limax in sage onto fresh wMY plates inoculated with a streak of both light and TEM morphology. However, strain NTSHR Escherichia coli (strain K-12 MG1655) as a food source. branches distantly away from S. limax sensu Amaral Zet- Cultures Nx13-1a and SK13-4e have been deposited with tler et al. 2000 (Dykova et al. 2008). This led Dykova et al. the ATCC under accession PRA-414 and PRA-415, respec- to recognize NTSHR as a representative strain of Sacc- tively. amoeba and that S. limax sensu Amaral Zettler et al. (2000) should be considered a distinct genus. However, Light microscopy they did not propose a name for that clade of strains. Although helical patterning of mitochondrial cristae is All light micrographs were taken using a Zeiss AxioSkop 2 observed in the morphologically similar genus Cashia (Carl Zeiss Microimaging, Thornwood, NY) connected to (Page, 1985) as noted by Dykova et al. 2008; the pattern- an 18-megapixel Canon 650D color digital camera (Canon, ing of Cashia is much less pronounced and less uniform Melville, NY). Image capture was performed by use of than in NTSHR (compare fig. 1E–H of Dykova et al. 2008 Canon’s image capture software (EOS Utility; Canon). to fig. 27 and 28 of Page 1985). Observations and photomicrographs using differential Here, we present three novel isolates of freshwater/soil interference contrast optics were made by inoculating a amoebae that were obtained from samples collected from drop of wMY liquid media with amoebae taken from agar eastern-central Mississippi. Our isolates have a general plates onto glass slides covered with a coverslip. Mea- appearance of saccamoebids, with limax-type morphology surements were taken using the measurement function of and frequently lack a distinctive hyaline cap. We present ImageJ (Schneider et al. 2012). morphological and ultrastructural data as well as an SSU rDNA phylogeny. One of our strains branches with the DNA extraction taxon “Saccamoeba limax” sensu Amaral Zettler et al. 2000 and LOS7N. Our other two strains are virtually identi- For DNA isolation, Nx13-1a, Nx13-1c, and Sk13-4e were cal to one another at the SSU rDNA level except for a cultured with E. coli (strain K-12 MG1655) on wMY agar 497-bp group I intron in strain Nx13-1c; together they plates at room temperature (ca. 21 °C). DNA was branch as sister to the amoebae with helical cristae, i.e. extracted from two plates of dense growth amoebae ATCC 30942 and LOS7N. using a modified salt extraction method (Aljanabi and Mar- tinez 1997) for use as a template in polymerase chain reaction (PCR) amplifications. Briefly, cells were resus- MATERIALS AND METHODS pended in 5-ml liquid wMY and collected in a 15-ml coni- cal tube. The cells were pelleted by centrifugation at Isolation and culturing 3184 g for 2 min, and the cell pellet was suspended in Strains Nx13-1a and Nx13-1c were isolated from a water 200 ll of salt homogenizing buffer (0.4 M NaCl, 10 mM sample that was collected from Sam D. Hamilton Noxu- Tris–HCl pH 8.0 and 2 mM ethylenediaminetetraacetic bee National Wildlife Refuge (33.273220°N, 88.790542°W) acid [EDTA] pH 8.0). Then proteinase K (20 mg/ml) was on September 2, 2013. The water sample was from water added to the solution to a final concentration of 400 lg/ml that was about 20-cm deep, with a mud sediment bottom. and sodium dodecyl sulfate was added to final concentra- Sediment was also obtained during the water collection. tion of 2%. The sample was mixed by gentle inverting Strain Sk13-4e was isolated from a soil sample that was and then incubated at 56 °C for 1 h. The sample was incu- collected in between the annex and lecture hall of Harned bated on ice for 5 min followed by the addition of one vol- Hall on the Mississippi State University campus, Missis- ume of 5M NaCl [2.5 M]. The sample was then vortexed sippi State, MS, USA (33.455790°N, 88.788123°W) on lightly and spun 14,000 g at 4 °C for 30 min. The superna- September 20, 2013. For the soil sample, as mud slurry tant was moved to a fresh tube and centrifuged again to was made with ~5 g of soil mixed with 15 ml of sterile pellet residual precipitant (14,000 g at 4 °C for 10 min). ddH2O. The slurry was then immediately used as an inoc- One volume of 100% isopropanol was added to each ulum. For the samples (water and soil/water slurry), three supernatant and inverted to mix. The tubes were water drops were placed onto the surface of three sterile incubated at 20 °C for 1 h and then spun at 14,000 g at wMY agar (0.002 yeast extract, 0.002 g malt extract, 4 °C for 30 min to precipitate the nucleic acid. To the

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 2 Journal of Eukaryotic Microbiology 2014, 0, 1–9 Watson et al. Ptolemeba n. gen., a New Hartmannellid Genus pellet, 1 ml of 75% Ethanol was added and spun at as Molecular phylogenetics above for 10 min. After drying, the pellet was rehydrated in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Twenty-nine publicly available SSU rDNA sequences were included with our novel sequences in our phylogenetic analyses. Our new sequences were manually added to an SSU rDNA sequencing existing seed alignment using Mesquite v2.75 (http:// The SSU rDNA from Nx13-1a and Nx13-1c was amplified mesquiteproject.org/), which was rich in Tubulinea, from using eukaryotic primers S1 (50-AACCTGGTTGATCCTGCC- Brown et al. (2011). Trees were inferred from an inclusion 30), and RibB (50-GATCCTTCTGCAGGTTCACC-30) (Fiore- set of 1,258 unambiguously aligned nucleotide positions Donno et al. 2008), GoTaq Green Master Mix (Promega, using maximum likelihood (ML) and Bayesian inference Madison, WI) following the manufacturer’s instructions, (BI) methods. A general-time-reversible + gamma distribu- and the following cycling parameters: 45 s initial denatur- tion + estimation of the proportion of invariant sites ation at 94 °C, followed by 33 cycles of 25 s denaturation (GTR+Γ+I) was implemented as suggested by the Akaike at 94 °C, 60 s annealing at 42 °C, and polymerization at Information Criterion in ModelTest v3.7 (Posada and Cran- 72 °C for 3.5 min. The PCR reactions were analyzed by dall 1998). The ML tree was estimated from 300 tree electrophoresis on 1% agarose gels using Tris-acetate searches implemented in RAxML v8.0 using 25 discrete (TA) gel and running buffer (4.84 Tris-base, 1.142 ml Gla- gamma rate categories (Stamatakis 2014). Topological cial Acetic Acid, filled to 1 liter with ddH2O). The SSU support was assessed by ML analyses of 1,000 bootstrap rDNA of Sk13-4e was amplified as above except with the replicates (MLBS) in RAxML. For BI, two independent primers 5017!SSU (50-CTGGTTGATCCTGCCAG-30) (Shad- runs consisting of four Markov Chain Monte Carlo were wick et al. 2009) and RiBb. For Nx13-1a and Nx13-1c, a run in MrBayes v3.12 (Ronquist and Huelsenbeck 2003) 1:100 dilution of the primary PCR reaction was used as a for 10,000,000 generations, sampling trees every 100 gen- template for nested secondary PCRs using the primer pair erations. The “burnin” was set to 2,500,000 generations, 30F (50-AAAGATTAAGCCATGCATG-30) (Shadwick et al. by which time all parameters converged as assessed by 2009) and 1492R (50-ACCTTGTTACGACTT-30) (Lane 1991) the “sump” function of MrBayes, an average standard and the above cycling parameters. For all strains, the SSU split deviation of < 0.0023, and the potential scale reduc- rDNA band (~1,800–2,100 bp) was cut out of the gel tion factor convergence diagnostic. under a UV-transilluminator and purified using a 200-ll bar- rier pipette tip trimmed to fit into a 1.5-ml microfuge tube. Transmission electron microscopy The agarose gel slice containing DNA amplicon was placed on top of the filter in the barrier pipette tip; the pip- For TEM, cells were grown on wMY plates and were har- ette tip was then put into a 1.5-ml tube and centrifuged at vested into a 15-ml conical tube in the same fashion as 17,900 g for 10 min to extract the liquid containing the DNA. The cells were centrifuged at 4,360 g for 2 min. The purified PCR product. By using TA as the gel buffer, which supernatant was poured off and the cell pellet suspended does not contain EDTA, the product was directly Sanger in a cocktail of culture media containing 2.5% v/v glutaral- sequenced. The PCR products were completely dehyde and 1% OsO4 and fixed for 30 min on ice. The sequenced using the PCR primers and internal sequencing fixed cells were then centrifuged at 4360 g for 1 min and primers. Microheterogeneity was assessed by the pres- the supernatant was removed. Cells were then washed ence of mixed peaks in chromatograms. The SSU rDNA once in liquid wMY medium. This process was repeated sequences are deposited in GenBank, accession numbers twice more, with H2O washes. Cells were again concen- KJ542108–KJ542110. trated by centrifugation, and then enrobed in 2.0% (w/v) agarose. Agarose blocks were dehydrated in a graded ser- ies of ethanols up to absolute ethanol, and then embed- Group I intron prediction ded in SPI-Pon 812 Epoxy resin (SPI Supplies, West A Group I catalytic intron in the SSU rDNA of Ptolemeba Chester, PA). Sections (50 nm) were cut with a diamond noxubium (isolate Nx13-1c) was first identified as a 497-bp knife on a Reichert-Jung Ultracut E Ultramicrotome, insertion in our SSU rDNA alignment. This intron corre- mounted on pioloform film in grids using the technique of sponds to position S516 in the E. coli 16s rRNA (GenBank Rowley and Moran (1975), and were subsequently stained accession AB03592), nomenclature of (Johansen and Hau- with saturated uranyl acetate in 50% ethanol and with gen 2001). The S516 intron was searched for similarity of lead citrate. Sections were observed using a JEOL 2100 predicted secondary structure to other Group I Intron 200 kV TEM electron microscope (JEOL USA, Peabody, sequences using the covariance model (CM) in the pack- MA). age Infernal v1.1 (http://infernal.janelia.org/) and a seed alignments from various subgroups of introns from Group RESULTS I Intron Sequence and Structure Database, the seed alignment of (http://www.rna.whu.edu.cn/data/alignment/) Light microscopy and TEM (Zhou et al. 2008). The secondary structure was further predicted using the RNAfold server (http://rna.tbi.univie.ac. Amoebae of Sk13-4e were predominantly monopodial li- at/) (Mathews et al. 2004) and is presented in Fig. S1. max (slug shaped) in appearance (Fig. 1). The lengths of

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–9 3 Ptolemeba n. gen., a New Hartmannellid Genus Watson et al.

Figure 1 Differential interference contrast light photomicrographs of Ptolemeba bulliensis n. gen. n. sp. amoebae, strain Sk13-4e. Scale bar = 10 lm.

Sk13-4e amoebae were 17.1–37.2 lm (mean = 25.6 lm, that is 2.6–5.5 lm in diam. (mean = 3.9 lm, SD = 0.73, SD = 4.2, n = 52), with breadths of 3.5–8.1 lm n = 22) with a prominent nucleolus that is 1.1–3.6 lm (mean = 5.5 lm, SD = 1.1, n = 52). The average length to (mean = 2.3 lm, SD = 0.66, n = 22) in diam. (Fig. 2A, B). breath ratio is 4.7 (Fig. 1). Cells possess a single nucleus There is sometimes a thin crescent-shaped hyaline cap on that is 2.4–4.8 lm in diam. (mean = 3.4 lm, SD = 0.57, the anterior end of amoebae, and on their posterior, there n = 44) with a prominent nucleolus that is 2.7–1.2 lm is a morulate-bulbous uroid (Fig. 2). Locomotive amoebae (mean = 1.9 lm, SD = 0.37, n = 44) in diam. (Fig. 1A, B, move in a monotactic fashion and can abruptly change E, G, H). Locomotive amoebae move in a monotactic fash- direction with semi-eruptive pseudopodial extensions ion and can abruptly change direction with semi-eruptive (Video S2). In locomotive forms, there is often a promi- pseudopodial extensions (Video S1). In locomotive forms, nent hyaline cap at the anterior end (Video S2). Over 7 mo there is often a prominent hyaline cap at the anterior end. of culture, no cysts were observed. This hyaline cap may however be absent (Video S1). On The mitochondrial cristal configuration varies between the posterior end, there is a villous-bulbous uroid (Fig. 1) strains Sk13-4e and Nx13-1a. Both strains have tubular (Video S1). Over 6 mo of culture, no cysts were observed. mitochondrial cristae, but Sk13-4e has a unique morphol- Amoebae of Nx13-1a and Nx13-1c were predominantly ogy where the tubular cristae are arranged in a uniform monopodial limax in appearance (Fig. 2). The lengths of fashion that appears to be a helical patterning (Fig. 3A, the amoebae were 11.9–29.8 lm (mean = 18.8 lm, S3). Strain Nx13-1a is much less uniform, with tubular SD = 3.62, n = 40), with breadths of 4.3–8.1 lm cristae that are not arranged in a helical pattern (Fig. 3B). (mean = 6.0 lm, SD = 0.96, n = 40). The mean length to Additional TEM micrographs of both taxa are available in breath ratio is 3.2 (Fig. 2). Cells possess a single nucleus Fig. S2–S5.

Figure 2 Differential interference contrast light photomicrographs Ptolemeba noxubium n. gen. n. sp. amoebae, strains Nx13-1a (A–D) and Nx13-1c (E–H). Scale bar = 10 lm.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 4 Journal of Eukaryotic Microbiology 2014, 0, 1–9 Watson et al. Ptolemeba n. gen., a New Hartmannellid Genus

Group I Intron in Nx13-1c As noted above, strain Nx13-1c has a 497-bp insertion that is unique from its sister strain Nx13-1a. The insertion sequence was searched for RNA secondary structural homology using a CM in Infernal, which yielded an expected value of 4.8e-37 to the IE3 subgroup of group I introns, suggesting that the secondary folding motif is highly similar to these introns. Subsequently, the second- Figure 3 Transmission electron micrographs of mitochondria in ary structure predicted in RNAfold of this group I intron is (A) Ptolemeba bulliensis n. gen. n. sp. strain Sk13-4e and (B) Ptolemeba available in Fig. S1. noxubium n. gen. n. sp. strain Nx13-1a. Scale bar = 200 nm.

DISCUSSION Trophozoites of the Hartmannellidae have a limax morphol- SSU rDNA data and phylogenetic analyses ogy that most often move in a monoaxial fashion (Page The SSU rDNA sequences obtained from our new strains 1988). Depending on the genus, they may or may not are 1,727, 2,226, and 1,927 bp, for Nx13-1a, Nx13-1c, and have a distinct hyaline cap or uroid and their nucleus mor- Sk13-4e, respectively. The 497 bp difference between phology is always vesicular (Page 1985). The Hartmannelli- strains Nx13-1c and Nx13-1a is attributed to the group I dae is historically a problematic taxonomic group within intron present in Nx13-1c (see below). Besides the group I the Tubulinea; because, their generic features make them intron, these strains differ by 2 bp (upd 0.00119, Table 1). difficult to assign a genus. Saccamoeba is a prime exam- Phylogenetic analyses of SSU rDNA data (Fig. 4) show ple of the problematic nature of hartmannellid taxonomy that strain Sk13-4e branches with full support with three (Smirnov et al. 2011). other publicly available SSU rDNA sequences attributed to Saccamoeba is a genus of amoebae found mainly in Saccamoeba (Amaral Zettler et al. 2000). Strain Sk13-4e is freshwater and soil. The amoebae are characterized by highly similar to sequences AF293902 (strain ATCC having a thin hyaline cap at the leading edge of an actively 30942) and AY145442 (strain LOS7N), with an upd of moving trophozoite. Saccamoeba was first described in 0.00645 and 0.00822, respectively, based on an alignment 1897 (Frenzel 1897), and later emended (Bovee 1972). In of 1,716 bp. These sequences are very similar to the SSU 1974, Page reevaluated the status of Saccamoeba noting rDNA sequence GU5690162 (strain T2), with an average that the original diagnosis of Saccamoeba “was so vague upd of 0.07. However, data from strain T2 is only available as to be of little use,” because the drawing could be as a truncated SSU rDNA sequence. Strains Nx13-1a and construed as any number of amoeboid taxa. Nonetheless, Nx13-1c are fully supported as the sister to the clade of our current concept of Saccamoeba is most adequately Sk13-4e + ATCC 30042 + LOS7N + T2 (Fig. 4). The maxi- diagnosed in Bovee 1972 (Page 1974). The genus consists mum upd in the clade is between Nx13-1c and T2, with of amoebae that often lack a hyaline cap, and have villous- an upd of 0.1977. The average upd between Sk13-4e and knob uroid and a vesicular nucleolus. Bovee considered strains NX13-1a and Nx13-1ac is 0.136 (Table 1). Our phy- the type species of the genus to be S. lucens (Bovee logenetic results show that these taxa branch robustly 1972; Page 1974). Of the available data on isolates within the Tubulinea clade (Fig. 4) and with high support attributed to the genus Saccamoeba, S. limax is probably (96% MLBS/1.0 BI) as sister to a clade of Amoeba + the most robust and therefore, stable (Page 1974). In Chaos + Copromyxa + Saccamoeba sensu stricto + Gla- accordance with the well-discussed argument in Dykova eseria (Fig. 4). Our phylogenetic analyses show that the et al. 2008; we recognize S. limax strain NTSHR clade that our strains are in is unique from the Sacc- (EU869301) as an exemplar strain of the genus amoeba clade, which is also fully supported (Fig. 4). Saccamoeba. Briefly, NTSHR has tubular mitochondrial

Table 1. Uncorrected pairwise distances between SSU rDNA sequences of the genus Ptolemeba

Taxon Strain Nx13-1a Nx13-1c Sk13-4e 30942 LOS7N

P. noxubium Nx13-1a – P. noxubium Nx13-1c 0.00119 – P. bulliensis Sk13-4e 0.13675 0.13555 – P. bulliensis 30942 0.13615 0.13615 0.00645 – P. bulliensis LOS7N 0.13793 0.13794 0.00822 0.00411 – P. sp. T2 0.19767 0.19770 0.06595 0.07442 0.07314

Pairwise distances among the coding regions of the SSU rDNA sequences were computed using an inclusion set of 1,716 nucleotide characters except for Ptolemeba sp., strain T2 where only a truncated sequence is available for analyses.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–9 5 Ptolemeba n. gen., a New Hartmannellid Genus Watson et al.

Figure 4 Maximum likelihood tree of 32 SSU rDNA sequences inferred under the GTR+I+Γ model inferred in RAxML. Support values at each node are presented for RAxML ML bootstrapping and Bayesian Inference posterior probabilities (MLBS/BI). Bootstrap values and BI posterior probabilities of 100%/1.0, respectively, are represented by a black dot. Support values less than 50% or 0.50 for the respective method are not presented. GenBank accession numbers of SSU sequences used in analyses are listed next to the taxon names. The scale bar represents evolu- tionary distance in changes per site. Cartoon schematics of mitochondrial cristal configurations for important taxa to this study are shown next to the taxa names. cristae that are irregularly patterned, whereas other strains is distinct from Glaeseria at the molecular level (Fig. 4) as attributed to Saccamoeba (i.e. LOS7N, sister to ATCC well as Glaeseria forms cyts. Unlike Glaeseria and Sacc- 30942) has tubular cristae arranged in a helical pattern (Dy- amoeba, there are no molecular data attributed to Cashia. kova et al. 2002). Further, given the size measurements However, our results suggest that Ptolemeba is a distinct of NTSHR, the isolate fits within the morphological spe- genus from Cashia. Cashia is reported to have unbranched cies concept of S. limax (Page 1974) and should be trea- helical mitochondrial cristae. Although our isolate Sk13-4e ted as such (Dykova et al. 2008). Moreover, the most has unbranched tubular mitochondrial cristae arranged in a recently described species of Saccamoeba, S. lacustris, uniform configuration like that of LOS7N (Dykova et al. falls within a clade with S. limax NTSHR (Corsaro et al. 2008), this configuration is dissimilar in pattern to that of 2010). Taking these points into account, the most appro- Cashia (see fig. 27 and 28 of Page 1985). Futhermore, priate taxonomic action is to accept the genus concept of Cashia does not form a villous-knob uroid structure (Page Saccamoeba sensu Dykova et al. (2008); and provide a 1988). Our strains Sk13-4e, Nx13-1a, and Nx13-1c, as well new genus concept for the clade of other amoebae previ- as LOS7N, almost always have a conspicuous villous knob ously recognized as Saccamoeba in SSU rDNA sequences, (Fig. 1, 2) (also see fig. 1B of Dykova et al. 2002). such as ATCC 30942 of Amaral Zettler et al. (2000). Here, Although the various strains of Ptolemeba represent sig- we name that clade as the new genus Ptolemeba n. gen., nificant genetic distances at the SSU rDNA level (average which herein contains two described species. intrageneric upd of 0.13), they form a fully supported clade Cashia and Glaeseria are other hartmannellid amoeba given the SSU rDNA data currently available (Fig. 4). genus that are morphologically similar to our Ptolemeba Genetic distances in other genera have been reported to be strains and Saccamoeba (Page 1985, 1988). For example, on the magnitude of our genus concept of Ptolemeba; for both Cashia and Glaeseria often lack a conspicuous hya- example, the heterolobosean genus Acrasis has upd of line cap similar to Ptolemeba and Saccamoeba. Ptolemeba greater than 0.15 (Brown et al. 2012). Moreover, these

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 6 Journal of Eukaryotic Microbiology 2014, 0, 1–9 Watson et al. Ptolemeba n. gen., a New Hartmannellid Genus strains of Ptolemeba are morphologically similar at the light Ptolemeba bulliensis Brown et al. n. sp microscopic level. Therefore, for name stability, we have decided to consider these taxa the same genus. Our results Diagnosis. Species with characteristics of the genus. suggest that there are at least two species, in the genus Freshwater inhabiting monotactic naked amoeba. Length Ptolemeba, Ptolemeba bulliensis n. sp. and P. noxubium n. in locomotion 25.6 lm (37.2–17.1), breadth 5.5 lm (L/B sp. The unpublished strain T2 (GU569162) probably repre- ratio: 4.7). Thin hyaline crescent-like cap at the anterior of sents another species, but unfortunately only a truncated the cell, and most often with a spineolate to villous- portion of the SSU rDNA sequence (241 bp) is available and bulbous uroid. Amoebae uninucleate with vesicular the ends are somewhat divergent from the other Ptole- nucleus 4.9–2.5 lm (mean = 3.4 lm) in diam., central meba species, which may be the result of sequencing error. nucleolus, 2.7–1.2 lm (mean = 1.9 lm) in diam. Mito- However, Ptolemba sp. strain T2 is almost certainly a chondria with straight, nonbranching, tubular cristae unique species and should be analyzed with full length SSU arranged in a uniform helical pattern. No distinct floating rDNA sequence data and morphological characterization. In form observed. No cyst observed. addition, it would be interesting to examine the mitochon- Type location. The type isolate of this species was iso- drial cristal configuration to see if it also possesses helical lated from topsoil collected in between the annex and lec- patterning, like P. bulliensis. It should be noted that mito- ture hall of Harned Hall on the Mississippi State University chondrial cristae might be a variable character in the hart- campus, Mississippi State, MS, USA (33.455790°N, mannellid amoebae. It is variable in the closely related 88.788123°W). euamoebid, Chaos carolinense, in which the cristal morpho- Type material. A fixed and embedded resin TEM block of logies changed from unbranched tubular to a cubic morphol- Sk13-4e was deposited in the Smithsonian Museum under ogy that could be interpreted as helical under starvation accession 1231517. This permanent physical specimen is conditions (Deng et al. 1999). More studies on cristal vari- considered the hapantotype (name-bearing type) of the ability should be conducted with the hartmannellid amoebae genus and species (see Art. 73.3 of the International Code to test for variability in this character. for Zoological Nomenclature, 4th Edition). The type culture Our data provide taxonomic clarification of the hartman- (Sk13-4e) has been deposited with the ATCC under acces- nellid genus Saccamoeba, which was previously shown to sion PRA-414. be paraphyletic (Brown et al. 2011; Corsaro et al. 2010; Dy- Gene sequence data. The nearly complete SSU rRNA kova et al. 2008; Smirnov et al. 2011). The data presented gene of the type isolate (Sk13-4e) is deposited in GenBank warrant the description a new genus that is morphologically under accession no. KJ542108. similar to Saccamoeba, but branches as sister to a clade Etymology. Similar to the genus’ etymology, the species that contains Saccamoeba sensu stricto in addition to other name bulliensis refers to the nickname of the mascot euamoebids (Amoeba, Chaos, Copromyxa, and Glaeseria). for Mississippi State University, “Bully,” spelled here as This study further illustrates the necessity of molecular data “bulli.” The species name is ended with “-ensis,” which in combination with morphological data to properly resolve means “from a place,” referring to the type locality. taxonomic issues with amoeboid protists. Ptolemeba noxubium Brown et al. n. sp TAXONOMIC SUMMARY Diagnosis. Species with characteristics of the genus. Amoebozoa Luhe 1913, emend. Cavalier-Smith 1998 Freshwater inhabiting monotactic naked amoeba. Length Tubulinea Smirnov et al. 2005 in locomotion 18.8 lm (29.8–12.0), breadth 6.0 lm (L/B Euamoebida Lepsi 1960, emend. Smirnov et al. 2011 ratio: 3.2). Thin hyaline crescent-like cap at the anterior of the cell, and frequently a villous-bulbous uroid that may be fasciculate. Amoebae uninucleate with vesicular nucleus Ptolemeba Brown et al. n. gen 5.5–2.6 lm (mean = 3.9) in diam., central nucleolus, 3.5– Diagnosis. Small bacterivorous, limax-shaped amoeba. 1.1 lm (mean = 2.2 lm) in diam. Mitochondria with tubu- Cells typically form broad lobose pseudopodia as wide as lar cristae. No distinct floating form observed. No cyst the cell proper often with a thin hyaline cap, but a promi- observed. nent hyaline cap may or may not be present. Locomotive Type location. The type isolate of this species was iso- trophozoites most commonly with a villous uroid. Mito- lated from freshwater/sediment sample collected from a chondrion with tubular cristae. Cysts not observed. bald cypress swamp, Bluff Lake in the Sam D. Hamilton Type species. Ptolemeba bulliensis n. sp. Noxubee National Wildlife Refuge (33.273220°N, Etymology. The genus name is derived from the name of 88.790542°W). the first mascot for Mississippi State University, a bulldog Type material. A fixed and embedded resin TEM block of named Ptolemy, who in turn was named after the Greco- the type isolate Nx13-1a was deposited in the Smithso- Roman mathematician, Claudius Ptolemy. We chose this nian Museum under accession 1231518. This permanent name because of type locality of the type isolate (Sk13- physical specimen is considered the hapantotype (name- 4e), which was the Mississippi State University campus. bearing type) of the species. The type culture (Nx13-1a) We chose to end the genus name with “-eba” to make has been deposited with the ATCC under accession the word sound like “amoeba.” PRA-415.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–9 7 Ptolemeba n. gen., a New Hartmannellid Genus Watson et al.

Gene sequence data. The nearly complete SSU rRNA Dykova, I., Veverkova, M., Fiala, I. & Machaekov a, B. 2002. A gene of the type isolate (Nx13-1a) is deposited in Gen- free-living amoeba with unusual pattern of mitochondrial struc- Bank under accession no. KJ542109. ture isolated from atlantic salmon, Salmo salar L. Acta Proto- – Etymology. The species name noxubium refers to the type zoologica, 41:415 419. locality of the species, the Sam D. Hamilton Noxubee Fiore-Donno, A. M., Meyer, M., Baldauf, S. L. & Pawlowski, J. 2008. Evolution of dark-spored Myxomycetes (slime-molds): mol- National Wildlife Refuge, often referred to as “Noxubee.” ecules versus morphology. Mol. Phylogenet. Evol., 46:878–889. Frenzel, J. 1897. Untersuchungen ueber die mikroscopische ACKNOWLEDGMENTS Fauna Argeniniens. Die Protozoen. I. Abt. Die Rhizopodien und Helioamoeben. Bibl. Zool., 12:1–82. This project includes independent undergraduate research Johansen, S. & Haugen, P. 2001. 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RNAfold predicted RNA secondary folding microscopic tomography. J. Struct. Biol., 127:231–239. Dykova, I., Kostka, M. & Peckova, H. 2008. Morphology and SSU structure of the predicted group I intron in Nx13-1c. The rDNA-based phylogeny of a new strain of Saccamoeba sp. figure depicts the minimum free energy (MFE) secondary (Saccamoeba Frenzel, 1892, Amoebozoa). Acta Protozoologica, structure with base-pair probabilities (color scale). The 47:397–405. minimum free energy is predicted as 122.60 kcal/mol.

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The RNAfold command line was “RNAfold -p -d2 –noLP < Figure S5. Transmission electron micrographs of Ptole- in > out.” meba noxubium n. sp. strain Nx13-1a showing mitochon- Figure S2. Whole cell section transmission electron dria (A) and the cell’s membrane (B). Scale bar of micrographs of Ptolemeba bulliensis n. gen. n. sp. strain A = 1 lm. Scale bar of B = 500 nm. Sk13-4e. Scale bar of A = 2 lm. Scale bar of B = 2 lm. Video S1. Real-time differential interference contrast Figure S3. Transmission electron micrographs Ptolemeba microscopical video of Ptolemeba bulliensis strain Sk13- bulliensis n. gen. n. sp. strain Sk13-4e showing mitochon- 4e. dria (A) and the cell’s membrane (B). Scale bar of Video S2. Real-time differential interference contrast A = 1 lm. Scale bar of B = 500 nm. microscopical video of Ptolemeba noxubium strain Nx13- Figure S4. Whole cell section transmission electron 1a and Nx13-1c. The first half of the video is strain Nx13- micrographs of Ptolemeba noxubium n. sp. strain Nx13- 1a and the latter half is Nx13-1c. 1a. Scale bar of A = 2 lm. Scale bar of B = 1 lm.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–9 9